Kenneth J. Broadley

The Langendorff heart preparation—Reappraisal of its role as a research and teaching model for coronary vasoactive drugs

Abstract The historical development and improvement of the Langendorff isolated perfused heart is traced with particular emphasis on its use for recording coronary vascular responses to drugs. A range of coronary vasoactive drugs are examined on a modified sensitive preparation. These drugs—catecholamines, 5-HT, histamine, acetylcholine, bradykinin, vasopressin, angiotensin, papaverine, sodium nitrite, and aminophylline—exhibited classical responses that compare favorably with those reported in the literature and with the in vivo responses. The actions of these drugs are reviewed and the advantages and limitations of the preparation as a teaching and research model are discussed.

SELECTIVE RESERPINE‐INDUCED SUPERSENSITIVITY OF THE POSITIVE INOTROPIC AND CHRONOTROPIC RESPONSES TO ISOPRENALINE AND SALBUTAMOL IN GUINEA‐PIG ISOLATED ATRIA

1 Dose‐response curves for the positive inotropic and chronotropic responses to isoprenaline were obtained in atria from untreated guinea‐pigs and those receiving various reserpine pretreatments. 2 Tension responses were unaffected, whereas rate responses were depressed by the lowest dose of reserpine (0.05 mg/kg i.p. at 24 hours). 3 With larger 24 h doses and a 3 day pretreatment, the rate and tension dose‐response curves were progressively displaced to the left, indicating supersensitivity which was greater for tension at each pretreatment. 4 No supersensitivity to histamine or Ca2+ could be detected, leading to the conclusion that it was selective for the β‐adrenoceptor agonists possibly at the receptor level. 5 As an indication of the adrenergic neurone depleting effectiveness of each reserpine dosage, preparations were exposed to test doses of β‐phenylethylamine. 6 Salbutamol was a partial agonist in untreated atria, the maximum rate (63.3%) and tension (10.0%) responses being less than those for isoprenaline. In atria from reserpine pretreated animals the supersensitivity was revealed as an increase of this maximum compared with isoprenaline. 7 The significance of this observation in relation to the possible mechanism of the supersensitivity is discussed.

THE ROLE OF H1 AND H2‐RECEPTORS IN THE CORONARY VASCULAR RESPONSE TO HISTAMINE OF ISOLATED PERFUSED HEARTS OF GUINEA‐PIGS AND RABBITS

1 The effects of histamine on the isolated perfused hearts of guinea‐pigs and rabbits were examined. Records of contractile force, heart rate and coronary perfusion pressure were obtained. 2 Histamine exerted positive inotropic and chronotropic effects which were antagonized by burimamide and attributed to stimulation of H2‐receptors. 3 The coronary vascular response to histamine differed between guinea‐pigs and rabbits. In guinea‐pig hearts, three phases were apparent: (a) An initial vasodilatation preceding any effects on heart force and rate was antagonized by mepyramine and therefore mediated by histamine H1‐receptors in the coronary circulation. (b) A secondary vasoconstriction was attributed to the increased myocardial compression during the positive inotropic and chronotropic responses. (c) The final, more predominant, component was a prolonged vasodilatation probably associated with the increased metabolic activity of the heart. 4 The latter two components were abolished together with the myocardial responses by burimamide. The remaining coronary vascular response was biphasic, consisting of a vasodilatation immediately followed by vasoconstriction. Both were antagonized by mepyramine and therefore mediated by H1‐receptors. 5 The coronary vascular response of rabbit hearts was similar but no direct vasodilatation was observed and it was concluded that histamine receptors in the coronary vasculature involve only vasoconstriction.

Characterization of β-adrenoceptors mediating relaxation of the guinea-pig ileum

Relaxation responses to sympathomimetic amines were recorded in potassium‐contracted segments of guinea‐pig ileum. Experiments were performed in the presence of phentolamine (5 times 10−6 m) to eliminate β‐adrenoceptor‐mediated effects. Metanephrine (10−5 m) and desmethylimipramine (5 times 10−7 m) were also present to prevent extraneuronal and neuronal uptake respectively. A potency order (‐)‐isoprenaline > (‐)‐noradrenaline > (‐)‐adrenaline was established, indicating a β1‐adrenoceptor involvement for this relaxation. The potency of salbutamol (β2‐selective) relative to isoprenaline in the ileum compared closely with its relative potency in isolated cardiac tissues (β1) but differed significantly from the value in lung parenchymal strips and vas deferens (β2). The pA2 values for antagonism of selective agonists (‐)‐noradrenaline (β1‐selective) and fenoterol (β2‐selective) by practolol (β1‐selective) were identical, indicating a single β1‐adrenoceptor population. The pA2 values for antagonism of these agonists by ICI 118,551 (β2‐selective) were also identical and compatible with a β1‐adrenoceptor population. Relaxation of the guinea‐pig ileum is therefore mediated via a homogeneous population of β1‐adrenoceptors.

Evidence from agonist and antagonist studies to suggest that the β1-adrenoceptors subserving the positive inotropic and chronotropic responses of the heart do not belong to two separate subgroups

The positive inotropic and chronotropic responses of guinea‐pig isolated left and right atria to 17 sympathomimetic amines were examined under conditions selected to control the pharmacological environment. Each agonist was compared with (‐)‐isoprenaline as the reference by constructing dose‐response curves. Equiactive molar concentration ratios relative to (‐)‐isoprenaline were calculated at the EC50 for rate and tension responses. Statistical analysis revealed that (‐)‐noradrenaline and (±)‐α‐methylnoradrenaline were tension selective whereas (±)‐α‐ethylisoprenaline and N‐methyldopamine were rate selective relative to (‐)‐isoprenaline. However, no structural trends emerged. The rank order of potency varied slightly between rate and tension, but an analysis of the regression and correlation coefficients indicated respectively that the equiactive molar concentration ratios and the rank orders could be considered identical on rate and tension. Antagonism of the (‐)‐isoprenaline‐induced rate and tension responses by acebutolol, atenolol, practolol and propranolol was assessed from the pA2 values which were almost identical for both parameters with each antagonist. It is concluded that the β1‐adrenoceptors mediating the positive inotropic and chronotropic responses do not warrant subdivision into two separate groups.

THE RELEASE OF A CORONARY VASODILATOR METABOLITE FROM THE GUINEA‐PIG ISOLATED PERFUSED HEART STIMULATED BY CATECHOLAMINES, HISTAMINE AND ELECTRICAL PACING AND BY EXPOSURE TO ANOXIA

1 A procedure involving two guinea‐pig isolated hearts perfused in series is described for detecting in the recipient heart the release of a possible coronary vasodilator metabolite from the donor heart. 2 Adrenaline and isoprenaline stimulated the rate and force of contraction and produced a multiphasic coronary vascular response, the predominant phase of which was vasodilatation. When the β‐adrenoceptors of the recipient heart were blocked, stimulation of the donor heart by the catecholamines was associated with a coronary vasodilatation of the recipient heart. 3 Histamine stimulated rate and force of contraction and was predominantly coronary vasodilator. After blockade of histamine H1‐and H2‐receptors in the recipient heart, coronary vasodilatation followed increases in activity of the donor heart in response to histamine. 4 These vasodilator responses of the recipient heart were attributed to the release from the donor heart of a vasodilator metabolite by the increased activity. This is the proposed mechanism for the predominant coronary vasodilator response to catecholamines and histamine. 5 Periods of electrically‐paced tachycardia and anoxia of the donor heart also led to the release of vasodilator activity. 6 The possible identity of the metabolite is discussed.

Functional antagonism as a means of determining dissociation constants and relative efficacies of sympathomimetic amines in guinea-pig isolated atria.

1 The positive inotropic and chronotropic responses to sympathomimetic amines were examined in guinea‐pig isolated atria. 2 The order of potency measured from EC50 values was isoprenaline > orciprenaline > salbutamol ≥ fenoterol > terbutaline. Terbutaline and salbutamol were partial agonists on rate and together with orciprenaline and fenoterol also on tension responses. 3 Functional antagonism by carbachol caused a rightwards shift of the dose‐response curve and depression of the maximum response. The rate maxima for orciprenaline, fenoterol and terbutaline were above that of isoprenaline. All the tension maxima were below isoprenaline. 4 Dissociation constants (KA) and relative efficacies (er) were determined by analogy with irreversible antagonism. 5 The relative orders of affinity (KA) were isoprenaline > orciprenaline > fenoterol > salbutamol > terbutaline. Affinities were identical on rate and tension. 6 The relative efficacies were all greater than isoprenaline for rate responses. On tension they were the same or less than isoprenaline. 7 The implications of these results are discussed, in particular the fact that a partial agonist has a greater efficacy than a full agonist.

Adenosine-induced bronchoconstriction of isolated lung and trachea from sensitized guinea-pigs

1 The bronchoconstriction of airway‐perfused lungs and contraction of superfused tracheal spirals from guinea‐pigs in response to adenosine were examined. 2 In lungs from untreated animals, adenosine had little effect unless the perfusion pressure was raised with carbachol (1.1 μm), when it caused a fall in perfusion pressure. However, if removed from guinea‐pigs sensitized with ovalbumen (5 mg and 10 mg i.p. 14 and 12 days before use), adenosine was bronchoconstrictor, exerting bronchodilator effects only at high (1 mg) doses. The constrictor response to adenosine (300 μg) was significantly greater than that in lungs from untreated or sham‐injected animals. 3 In superfused trachea from untreated animals, adenosine exerted only relaxant responses. In tissues from ovalbumen‐sensitized guinea‐pigs adenosine produced contracile responses, with relaxation appearing only at high (1 mg) doses. 4 Thus sensitization by antigen challenge revealed a bronchoconstrictor response of isolated airway preparations to adenosine. This is related to the clinical situation where only asthmatic subjects respond to adenosine by bronchoconstriction and suggests that the sensitization may destabilize inflammatory cells for mediator release by adenosine. 5 The response to a second exposure to adenosine was consistently reduced (lungs) or converted to a relaxation (trachea) indicating tachyphylaxis and consistent with a mediator release mechanism. 6 The P1‐purinoceptor antagonist, 8‐phenyltheophylline (3.9 μm), antagonized the relaxant responses to higher doses of adenosine. However, it did not affect the contractile responses to lower doses of adenosine. Whether this is due to P1‐purinoceptors not being involved in the contractile response, or whether preferential blockade of the relaxant response leaves the contraction unopposed and apparently unblocked, remains to be established.

Cardiac alpha- and beta-adrenoceptor sensitivity and binding characteristics after chronic reserpine pretreatment

SummaryCardiac alpha- and beta-adrenoceptor sensitivities were examined after chronic pretreatment of rats with reserpine. Increases in sensitivity would indicate that the receptor is under the influence of the sympathetic innervation, removal by catecholamine depletion with reserpine of the tonic effect of neurotransmitter release would permit receptor upregulation. The positive inotropic responses of paced left atria and papillary muscles and the positive chronotropic responses of spontaneously beating right atria were recorded. A concentration-response curve to isoprenaline (beta-adrenoceptor-mediated) was followed, in the presence of beta-blockade, by one to methoxamine (alpha-adrenoceptor-mediated). Methoxamine exerted positive inotropy of left atria and papillary muscles, the maxima being 43.2 ± 2.7 and 26.8 ± 4.4% of the isoprenaline maxima. A small positive chronotropy (16.5 ± 5.6% maximum) of right atria occurred. After pretreatment with reserpine (1.0 mg kg−1 i.p. daily) for 7 days, the three preparations displayed supersensitivity to isoprenaline, revealed as a significant displacement (P < 0.05) of the concentration-response curves to the left of those for control rats. Reserpine pretreatment, however, had no effect on the sensitivity to methoxamine. The increase in beta-adrenoceptor sensitivity to isoprenaline after reserpine pretreatment was accompanied by a significant 41.3% increase (P < 0.05) in the number of [3H]-dihydroalprenolol ([3H]-DHA) binding sites (Bmax) in ventricular membranes, although the dissociation constant (KD) was unaffected. There were more alpha-adrenoceptor [3H]-prazosin binding sites in ventricular than atrial membranes. However, there was no difference in KD or Bmax between reserpine-pretreated and control tissues. It is proposed that the increase in beta-adrenoceptor sensitivity and binding sites arises from the depletion-induced loss of neuronal noradrenaline release onto the beta-adrenoceptors which are therefore directly under the influence of the neurotransmitter. The failure of cardiac alpha-adrenoceptors to exhibit supersensitivity and increased numbers suggests that they are not directly affected by the sympathetic innervation.

β-Adrenoceptor ligand binding and supersensitivity to isoprenaline of ventricular muscle after chronic reserpine pretreatment

Summary1.Isolate papillary muscles from guinea-pig hearts were paced at a constant frequency and isometric contractions recorded.2.Guinea-pigs were either untreated or pretreated with reserpine. Three pretreatment schedules were used; a) 0.5 mg kg−1 i.p. at 24h, b) 5.0 mg kg−1 at 72h and 3.0 mg kg−1 at 48 and 24 h, or c) 0.1 mg kg−1 daily for 7 days.3.Cumulative concentration-response curves for the isoprenaline-induced increases in tension were obtained. The geometric mean EC50 values after the 3 and 7 day reserpine pretreatment schedules were significantly (P<0.05) less than for untreated guinea-pigs indicating a supersensitivity.4.EC50 values for the positive inotropic inotropic responses to histamine and calcium in papillary muscles from reserpine-pretreated guinea-pigs did not differ significantly (P<0.05) from those from untreated animals. This suggests that the supersensitivity to isoprenaline is β-adrenoceptor specific.5.Membrane fractions were prepared from the ventricles of the untreated and reserpine-pretreated guinea-pigs from which papillary muscles had been removed. Binding of [3H]-dihydroalprenolol ([3H]-DHA) to β-adrenoceptors of these membranes was determined. Equilibrium dissociation constants (KD) and total numbers of binding sites (Bmax) were determined by Scatchard analysis of the saturation curves for [3H]-DHA binding.6.There was no increase in affinity (fall in KD value) or change in the total number of binding sites associated with reserpine-induced supersensitivity. The equilibrium inhibition constant (Ki) for the displacement of [3H]-DHA binding by isoprenaline was also identical in membranes from untreated and reserpine-pretreated animals. Thus reserpine-induced supersensitivity to isoprenaline does not appear to involve a change in affinity for the β-adrenoceptor or in receptor numbers as determined by [3H]-DHA binding.